Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “HEPES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32Linked to original sources
PubMed1992

Studies of transepithelial Cl- transport in cultured cauda epididymal cells of rats by the short-circuit current method.

1. Monolayer cultures of cauda epididymides from male Sprague-Dawley rats (210-230 g) were studied by the short-circuit current (ISC) technique to characterize the properties of the transepithelial chloride transport. In HCO(3-)-free, HEPES-buffered solution, adrenaline (0.23 microM) added to the basolateral side led to an increase in ISC and transepithelial conductance (gt). 2. Decreasing apical chloride concentration ([Cl-]a) progressively from 126.7 to 0 mM by substituting chloride with gluconate increased the ISC response to adrenaline (delta ISC) in a linear fashion with a slope of -1.6 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. Pretreating the tissue with a chloride channel blocker diphenylamine-2-carboxylate (DPC) on the apical side significantly reduced the slope to -4.9 x 10(-4) mu equiv h-1 cm-2 per millimolar change in [Cl-]a. 3. By substituting apical chloride with various anions and measuring the change in ISC upon adrenaline stimulation, the selectivity sequence of the apical anion conductance was found to be NO3- approximately Br- > Cl- > I- > gluconate > isethionate. 4. When the monolayers were bathed with Krebs-Henseleit solution containing 25 mM HCO3- and 5% CO2, the delta ISC at each [Cl-]a as well as the dependence of delta ISC on [Cl-]a (slope = -3.3 x 10(-3) mu equiv h-1 cm-2 per millimolar change in [Cl-]a) were significantly greater than the HCO(3-)-free counterpart. Addition of 0.1 mM acetazolamide or 0.5 mM SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) to the basolateral side significantly reduced the effects of HCO3- and CO2. 5. When the tissues were bathed on both sides with HCO(3-)-free, HEPES-buffered solution and were clamped at various transepithelial potential differences (PDt) from +30 mV (lumen positive) to -30 mV (lumen negative), the relationship between the clamping current response to adrenaline (delta ICL) and the PDt applied was linear. Zero clamping current response was found at -6 mV. Decreasing [Cl-]a to 0 mM reduced the dependence of delta ICL on PDt and delta ICL was positive at all PDt tested. The response of the transepithelial conductance to adrenaline (delta gt) did not depend on the PDt applied but was reduced with decreasing apical chloride concentration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Coupling of dual acid extrusion in the guinea-pig isolated ventricular myocyte to alpha 1- and beta-adrenoceptors.

1. Intracellular pH (pHi) was recorded in single, isolated guinea-pig ventricular myocytes using the pH-sensitive fluorophore, carboxy-SNARF-1 (AM-loaded). 2. The dual acid extrusion system in this cell (Na(+)-H+ antiport and Na(+)-HCO3- symport) was activated by inducing an intracellular acid load, produced by addition and subsequent removal of extracellular 10 mM NH4Cl. Under these conditions, it is known that both acid-equivalent extruders are activated about equally. 3. Application of phenylephrine (100 microM; alpha-adrenergic agonist) resulted in an inhibition of pHi recovery from an acid load, recorded in HCO3-buffered medium containing 1.5 mM amiloride (amiloride inhibits Na(+)-H+ antiport; under these conditions pHi recovery is mediated through only the Na(+)-HCO3- symport carrier). This inhibitory effect of phenylephrine was prevented by the alpha 1-antagonist, prazosin (0.1 microM) and was unaffected by propranolol (1 microM). 4. Application of phenylephrine in Hepes-buffered medium (only Na(+)-H+ antiport is active under these conditions) elicited a stimulation of pHi recovery, again prevented by prazosin (0.1 microM). 5. These results point to an alpha 1 inhibition of Na(+)-HCO3- symport and an alpha 1 stimulation of Na+-H+ antiport. 6. Both adrenaline (1-5 microM) and noradrenaline (5 microM) slowed pHi recovery recorded in HCO3(-)-buffered solution containing amiloride (1.5 mM). The similarity of this result with that obtained previously using phenylephrine (paragraph 3) suggests that all three agonists inhibit the Na(+)-HCO3- symport through alpha 1 activation. 7. Isoprenaline (1 microM; beta-adrenergic agonist) slowed pHi recovery in Hepes-buffered solution but stimulated recovery in a HCO3(-)-buffered solution containing amiloride (1.5 mM). These results suggest that beta activation slows Na(+)-H+ antiport but stimulates Na(+)-HCO3- symport. 8. When both acid-equivalent extrusion carriers were inhibited in Na(+)-free, HCO3(-)-buffered medium, phenylephrine or isoprenaline had no effect on pHi, ruling out any effect of the adrenergic agonists on background acid-loading mechanisms. 9. Under physiological conditions (CO2/HCO3(-)-buffered solution, no amiloride), when both acid extruders would be activated by an intracellular acid load, application of phenylephrine, adrenaline or noradrenaline were found to slow pHi recovery. In contrast, isoprenaline stimulated pHi recovery under the same conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenergic alpha-Agonists↗
PubMed1996

Effects of extracellular pH on voltage-gated Na+, K+ and Ca2+ currents in isolated rat CA1 neurons.

1. The effects of extracellular H+ (pHo) in the pathophysiological range (pH 6-8) on voltage-gated sodium, potassium, and calcium currents were examined in acutely dissociated rat hippocampal CA1 neurons using the whole-cell patch clamp technique. All experiments were conducted in Hepes-buffered solutions and were performed at room temperature (21-23 degrees C). 2. TTX-sensitive sodium currents, evoked by both step and ramp depolarization, were reversibly depressed by moderate acidosis and enhanced slightly by alkaline exposure. Changes in current amplitude were coincident with small reversible shifts (+/- 3 mV) in the voltage dependence of activation. In contrast, sodium current activation and decay kinetics as well as steady-state inactivation were unaffected by acidosis. 3. Outward potassium currents could be separated into a transient, rapidly inactivating current (IA) and a sustained, slowly inactivating component (IK). Steady-state activation of both currents was unaffected by an increase or decrease in pHo. Similarly, IK activation and IA decay kinetics remained stable during pHo exchange. In contrast, the steady-state inactivation (h infinity) of both potassium currents was reversibly shifted by approximately +10 mV during acid exposure, but remained unchanged during alkaline treatment. 4. Calcium currents were found to be predominantly of the high-voltage-activated (HVA) type, which could be carried by Ba2+ and inhibited completely by cadmium. Moderate acidosis (pH 6.9-6.0) reversibly depressed HVA Ca2+ current amplitude and caused a positive shift in its voltage dependence. For both of these parameters, alkaline treatment (pH 8.0) had the opposite effect. The depression of HVA Ca2+ currents by low pHo was unaffected by raising the internal Hepes concentration from 10 to 50 mM in the patch pipette. A Hill plot of the effect of pH on Ca2+ current amplitude revealed a pK value (defined as the mid-point of the titration curve) of 7.1 and a slope of 0.6. 5. The rate of Ca2+ current activation was unaffected by pHo at positive potentials, but below 0 mV the activation rate increased at low pH and decreased at high pH, becoming significant at -20 mV. At this membrane voltage, a second HVA current was revealed during acid exposure as the whole-cell HVA current was depressed. Ca2+ current decay was described by two time constants, both of which were significantly reduced at pH 6.4 and slightly enhanced at pH 8.0. Steady-state Ca2+ current inactivation reached 50% near -50 mV and was not affected at either pH extreme. 6. These results demonstrate that extracellular pH shifts within the pathophysiological range are capable of modulating both the conductance and gating properties of voltage-gated ion channels in hippocampal CA1 neurons. The effects we describe are consistent with the wellknown effects of pHo on neuronal excitability and strengthen the idea that endogenous pHo shifts may help regulate cell activity in situ.

Animals↗
PubMed2006

Transporters involved in regulation of intracellular pH in primary cultured rat brain endothelial cells.

Fluid secretion across the blood-brain barrier, critical for maintaining the correct fluid balance in the brain, entails net secretion of HCO(3)(-), which is brought about by the combined activities of ion transporters situated in brain microvessels. These same transporters will concomitantly influence intracellular pH (pH(i)). To analyse the transporters that may be involved in the maintenance of pH(i) and hence secretion of HCO(3)(-), we have loaded primary cultured endothelial cells derived from rat brain microvessels with the pH indicator BCECF and suspended them in standard NaCl solutions buffered with Hepes or Hepes plus 5% CO(2)/HCO(3)(-). pH(i) in the standard solutions showed a slow acidification over at least 30 min, the rate being less in the presence of HCO(3)(-) than in its absence. However, after accounting for the difference in buffering, the net rates of acid loading with and without HCO(3)(-) were similar. In the nominal absence of HCO(3)(-) the rate of acid loading was increased equally by removal of external Na(+) or by inhibition of Na(+)/H(+) exchange by ethylisopropylamiloride (EIPA). By contrast, in the presence of HCO(3)(-) the increase in the rate of acid loading when Na(+) was removed was much larger and the rate was then also significantly greater than the rate observed in the absence of both Na(+) and HCO(3)(-). Removal of Cl(-) in the presence of HCO(3)(-) produced an alkalinization followed by a resumption of the slow acid gain. Removal of Na(+) following removal of Cl(-) increased the rate of acid gain. In the presence of HCO(3)(-) and initial presence of Na(+) and Cl(-), DIDS inhibited the changes in pH(i) produced by removal of either Na(+) or Cl(-). These are the expected results if these cells possess an AE-like Cl(-)/HCO(3)(-) exchanger, a 'channel-like' permeability allowing slow influx of acid (or efflux of HCO(3)(-)), a NBC-like Cl(-)-independent Na(+)-HCO(3)(-) cotransporter, and a NHE-like Na(+)/H(+) exchanger. The in vitro rates of HCO(3)(-) loading via the Na(+)-HCO(3)(-) cotransporter could, if the transporter is located on the apical, blood-facing side of the cells, account for the net secretion of HCO(3)(-) into the brain.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1980

Plaquing procedure for infectious hematopoietic necrosis virus.

A single overlay plaque assay was designed and evaluated for infectious hematopoietic necrosis virus. Epithelioma papillosum carpio cells were grown in normal atmosphere with tris(hydroxymethyl)aminomethane- or HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid)-buffered media. Plaques were larger and formed more quickly on 1- to 3-day-old cell monolayers than on older monolayers. Cell culture medium with a 10% addition of fetal calf serum (MEM 10) or without serum (MEM 0) were the most efficient virus diluents. Dilution with phosphate-buffered saline, saline, normal broth, or deionized water reduced plaque numbers. Variations in the pH (7.0 to 8.0) of a MEM 0 diluent did not affect plaque numbers. Increasing the volume of viral inoculum above 0.15 ml (15- by 60-mm plate) decreased plaquing efficiency. Significantly more plaques occurred under gum tragacanth and methylcellulose than under agar or agarose overlays. Varying the pH (6.8 to 7.4) of methylcellulose overlays did not significantly change plaque numbers. More plaques formed under the thicker overlays of both methylcellulose and gum tragacanth. Tris(hydroxymethyl)aminomethane and HEPES performed equally well, buffering either medium or overlay. Plaque numbers were reduced when cells were rinsed after virus adsorption or less than 1 h was allowed for adsorption. Variation in adsorption time between 60 and 180 min did not change plaque numbers. The mean plaque formation time was 7 days at 16 degrees C. The viral dose response was linear when the standardized assay was used.

Adsorption↗
PubMed1989

Cadmium Resistance Screening in Nitrilotriacetate-Buffered Minimal Media.

Media used to determine the MICs of heavy metals for bacteria are unreliable because organic components in the media bind or chelate most of the metal being studied. To define specific metal activity in media and to maintain metal activity at a constant level, HEPES-MES [N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid-2-(N-morpholine)ethanesulfonic acid] salts medium with arabinose medium was modified, and the modified medium was used to examine the MIC of cadmium for Rhizobium fredii USDA 201. Arabinose-HEPES-MES was modified by addition of the chelator nitrilotriacetate to buffer the supply of free Cd ion to maintain a constant Cd activity and by the use of only MES to buffer pH (buffered arabinose-MES medium [BAM]). Ca and Mg were supplied at the normal levels for soil solutions, and other trace elements were supplied at the levels required for normal growth of plants. The concentration of free Cd ion was calculated by using the computer program GEOCHEM-PC with a corrected data base. The Cd MIC in BAM was 14.0 muM, while that in a tryptone-yeast extract medium was 107 muM. The results indicate that substantial free Cd is removed from solution in most standard media, resulting in falsely high MICs. The new BAM medium allows for the precise determination of MICs, thus avoiding the uncertainties associated with other media.

Journal Article↗
PubMed1995

Effects of select medium supplements on in vitro development of Cryptosporidium parvum in HCT-8 cells.

Surface-sterilized oocysts of Cryptosporidium parvum were applied to subconfluent monolayers of human adenocarcinoma (HCT-8) cells grown on coverslips in six-well cluster plates. Parasite-infected cultures were then incubated in RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer, and antibiotics at 37 degrees C in a 5% CO2-95% air incubator for 2 h to allow sporozoites to excyst and enter cells. After cultures were washed free of debris, fresh cell culture media containing select supplements were added and cultures were reincubated. Parasite growth was assessed 66 h later by counting the number of parasite developmental stages in 25 random x 100 oil fields by Nomarski interference-contrast microscopy. Four vitamin supplements, calcium pantothenate, L-ascorbic acid, folic acid, and 4-(para)-aminobenzoic acid, each resulted in a significant increase in parasite numbers in vitro. The addition of insulin and the sugars glucose, galactose, and maltose also had a positive effect on parasite growth, although the effect was less pronounced than with any of the vitamins. Using the above information, we developed a supplemental medium formulation consisting of RPMI 1640 with 10% fetal bovine serum, 15 mM HEPES, 50 mM glucose, and 35 micrograms of ascorbic acid, 1.0 micrograms of folic acid, 4.0 micrograms of 4-aminobenzoic acid, 2.0 micrograms of calcium pantothenate, 0.1 U of insulin, 100 U of penicillin G, 100 micrograms of streptomycin, and 0.25 microgram of amphotericin B (Fungizone) per ml (pH 7.4). The growth of c. parvum in this medium was found to be enhanced approximately 10-fold compared with that in control medium without additional glucose, insulin, or vitamins.

Animals↗
PubMedDate not supplied

Regulation of cell volume and intracellular pH in hyposmotically swollen rat osteosarcoma cells.

The maintenance of cell volume involves transduction of a volume-sensing signal into effectors of volume-regulatory transporters. After exposure to anisotonic conditions, cells undergo compensatory volume changes that are mediated by active transport and passive movement of ions and solutes. Intracellular pH (pHi) homeostasis may be compromised during these processes. We have studied pHi and some of the signal transduction mechanisms involved in the regulatory volume decrease (RVD) that occurs after exposure to hypoosmolar conditions in rat osteosarcoma cells, ROS 17/2.8. Cells were loaded with BCECF; pHi and cell volume were estimated by dual excitation ratio fluorimetry. Swelling of cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered hypotonic medium induced a rapid cell swelling followed by an incomplete RVD of approximately 30% in suspended (i.e., round) cells and approximately 60% in attached (i.e., spread) cells that was independent of subpassage number. RVD was inhibited by ouabain, valinomycin, and high external [K+], all of which should reduce the cell membrane electrochemical gradient for K+. Inhibition of RVD was induced also by decreasing intracellular [Ca2+] with BAPTA-AM and by depletion of Cl-, indicating the role of calcium-regulated K+ and Cl- efflux during RVD. Depolymerization of actin filaments by cytochalasin D prolonged the RVD three-fold and nonspecific activation of GTP-binding proteins up-regulated RVD. In attached cells the hypoosmolar-induced swelling caused a large reduction in pHi (approximately 0.7 units), which was sustained as long as cells were in hypoosmotic medium. The reduction of pHi induced by cell swelling was inhibited by Na(+)-free extracellular medium, ouabain, the tyrosine kinase inhibitor genistein, and to a lesser extent by Cl(-)-free medium. However, amiloride failed to inhibit the hypoosmolar-induced reduction of pHi. Collectively these data indicate that RVD of ROS 17/2.8 cells in HEPES-buffered medium is dependent on conductive efflux of K+ and Cl- that is regulated by cell shape, actin, and GTP-binding proteins. The sustained inhibition of pHi homeostasis induced by cell swelling may reflect the existence of cell volume sensing mechanisms that operate through tyrosine kinases to regulate pHi.

Actins↗
PubMed1977

An analysis of the action of lanthanum on aortic tissue from normotensive and spontaneously hypertensive rats.

Significant tension development resulting from the administration of lanthanum salts (La3+) to isolated aorta tissues was observed in preparations from the spontaneously hypertensive rat (SHR) but not in preparation from age-matched normotensive Wistar rats (NWR). The response to La3+ was significantly greater in tissues maintained in bicarbonate-buffered Krebs than observed in bicarbonate and phosphate-free HEPES-buffered Krebs. At least part of the LA3+ response in the bicarbonate- and phosphate-buffered solutions was due to a pH shift and could be mimicked by raising the extracellular hydrogen ion [H+] concentration. However, a direct action of La3+ on excitation-contraction coupling could be observed in HEPES-buffered solutions where the addition of La3+ also resulted in tension development but no significant pH change; this action of La3+ was found to be resistant to inhibition by maintenance in a Ca2+-free medium and (or) D-600 pretreatment suggesting an intracellular action for La3+. The paradoxical response to both H+ and La3+ in the SHR aorta suggests that the muscle membrane may be more permeable to these ions and, in addition, suggests that the membrane and (or) intracellular calcium stores in this tissue may also be more labile than those in the normotensive controls.

Animals↗
PubMed1991

Free radicals may contribute to oxidative skeletal muscle fatigue.

We used mouse soleus in vitro (n = 30) and canine gastrocnemius-plantaris preparations (n = 20) pump-perfused at the animal's blood pressure to establish if free radicals contribute to fatigue in oxidative skeletal muscle. The soleus from each leg contracted for 200 ms (70 Hz) once every minute for 60 min in Hepes buffer gassed with 100% oxygen at 27 degrees C. When contracting in Hepes alone, both muscles fatigued at 0.9 mN/mm2.min over the 60 min. The addition of purines to the bath increased the rate to 1.4 mN/mm2.min and the addition of xanthine oxidase to generate free radicals increased the rate again to 1.9 mN/mm2.min. Thus free radicals appeared to attenuate oxidative skeletal muscle function. Each canine muscle contracted isometrically at 4 Hz for 30 min and then rested for 45 min before contracting for a second 30 min at 4 Hz. In each experiment, we infused saline at 0.76 mL/min into resting muscle and at 1.91 mL/min during the first contraction period. During the remainder of the experiment, we infused, at the same rates, saline (n = 4), 10 microM dimethyl sulfoxide (DMSO) (n = 4) to identify the effect of scavenging hydroxyl radicals, 1 mM allopurinol to establish the effect of blocking xanthine oxidase (n = 4), or 200 microM desferoxamine to determine the effect of chelating iron (n = 4). With saline, the fatigue rate over the 30 min of contractions increased from 5.0 +/- 0.2 to 6.3 +/- 0.5 N/kg.min from the first to the second stimulation period. The fatigue rate was slower in the second period with each of the three experimental substances (DMSO, 5.9 +/- 0.8 to 3.2 +/- 0.3; allopurinol, 7.3 +/- 1.1 to 4.6 +/- 0.6; desferoxamine, 6.8 +/- 0.8 to 4.4 +/- 0.8 N/kg.min). The fatigue rate was the same as control when DMSO was infused only during the second contraction period. Therefore, free radicals appeared to contribute to fatigue in oxidative skeletal muscle.

Animals↗
PubMed1988

pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3-.

We have developed a technique to measure the fluorescence of a pH-sensitive dye (2,7-biscarboxyethyl-5(6)-carboxyfluorescein) in single glomerular mesangial cells in culture. The intracellular fluorescence excitation ratio of the dye was calibrated using the nigericin-high-K+ approach. In the absence of CO2-HCO3-, mesangial cells that are acid loaded by an NH+4 prepulse exhibit a spontaneous intracellular pH (pHi) recovery that is blocked either by ethylisopropylamiloride (EIPA) or removal of external Na+. This pHi recovery most probably reflects the activity of a Na+-H+ exchanger. When the cells are switched from a N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered solution to one containing CO2-HCO3-, there is an abrupt acidification due to CO2 entry, which is followed by a spontaneous recovery of pHi to a steady-state value higher than that prevailing in HEPES. Both the rate of recovery and the higher steady-state pHi imply that the application of CO2-HCO3- introduces an increase in net acid extrusion from the cell. One third of total net acid extrusion in CO2-HCO3- is EIPA sensitive and most likely is mediated by the Na+-H+ exchanger. The remaining two thirds of acid extrusion could be caused by a decrease in the background acid-loading rate and/or the introduction of a new, HCO3- -dependent acid-extrusion mechanism. The HCO3- -induced alkalinization cannot be accounted for by a HCO3- -induced reduction in the acid-loading rate. The latter can be estimated by applying EIPA in the absence of HCO3- and observing the rate of pHi decline. We found that this acid-loading rate is only about one fifth as great as the total net acid extrusion rate in the presence of HCO3-. Indeed, two thirds of net acid extrusion in HCO3- is blocked by 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), an inhibitor of HCO3- -dependent transport. Furthermore, the effects of EIPA and SITS were additive. Thus, in the presence of CO2-HCO3-, a SITS-sensitive-HCO3- -dependent transporter is the dominant mechanism of acid extrusion. This mechanism also accounts for the increase in steady-state pHi on addition of CO2-HCO3-.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Reduction in sensitivity to Cl- channel blockers by HCO3- -CO2 in rabbit cortical collecting duct.

We examined the ability of HCO3- -CO2 to modify the potency of Cl- channel blockers in the renal cortical collecting duct (CCD) for the following two reasons. 1) From a practical point of view, there is, to our knowledge, no information regarding the effect of the HCO3- -CO2 buffer system on the potency of Cl- channel blockers. 2) We showed in the companion manuscript [Am. J. Physiol. 257 (Cell Physiol. 26): C94-C101, 1989] that HCO3- -CO2 stimulates transepithelial anion exchange in the CCD. Based on precedent in the literature, we postulated that HCO3- stimulates the basolateral membrane Cl- conductance. Here, we demonstrate that several Cl- channel blockers can reduce CCD Cl- self exchange when the solutions are buffered in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES). Concentrations of blockers producing 80% inhibition in HEPES, pH 7.4, produced only 20% inhibition in 25 mM HCO3- -CO2, pH 7.4. The ability of HCO3- -CO2 to reduce blocker potency had an IC50 of only 2 mM. We also examined interactions of HCO3- -CO2 and blockers with regard to the principal cell basolateral Cl- conductance. Blockers did not alter the Rb+ flux, a marker of K+ transport, but did reduce transepithelial conductance (GT), i.e., the blockers inhibited the principal cell basolateral Cl- conductance. As was the case with intercalated cell anion exchange, GT measurements indicated that HCO3- -CO2 impaired the ability of Cl- channel blockers to inhibit the principal cell Cl- conductance.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed1989

Sodium and chloride transport across the isolated porcine gallbladder.

Porcine gallbladder, stripped of serosal muscle, mounted in Ussing chambers, and bathed in plasma-like Ringer solution generates a serosal positive transepithelial potential of 4-7 mV and a short-circuit current (Isc) of 50-120 microA/cm2. Substitution of Cl with gluconate or HCO3 with N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) results in a 50% decrease in Isc. Treatment with 1 mM amiloride (mucosal side) or 0.1 mM acetazolamide (both sides) causes 25-27% inhibition of the Isc. Mucosal addition of 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid inhibits the Isc by 17%. Serosal addition of 0.1 mM bumetanide inhibits the Isc by 28%. Amiloride (1 mM) inhibits the net transepithelial fluxes of Na and Cl by 55 and 41%, respectively. Substitution of Cl with gluconate inhibits the net Na flux by 50%, whereas substitution of HCO3 with HEPES inhibits 85-90% of the net Na flux and changes Cl absorption to net secretion. Based on these results, it is hypothesized that Na and Cl transport across the apical membrane is mediated by two pathways, Na-H/Cl-HCO3 exchange and Na-HCO3 cotransport. Partial recycling of Cl and HCO3 presumably occurs through a Cl conductive pathway and Cl-HCO3 exchange, respectively, in the apical membrane. This results in net Na absorption, which accounts for most of the Isc observed under basal conditions. The effect of bumetanide on the basolateral membrane and the fact that Cl secretion occurs when HCO3 is absent suggests that Cl secretion involves a basolateral NaCl or Na-K-Cl cotransport system arranged in series with a Cl conductive pathway in the apical membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1993

Amiloride-sensitive sodium conductance in human B lymphoid cells.

The Na+ conductance of RPMI 8226 human B lymphoblastoid cells was examined using whole cell patch clamp. When the bath solution contained RPMI 1640 and the pipette solution contained (in mM) 100 potassium gluconate, 30 KCl, 10 NaCl, 0.5 ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), and 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), as well as < 10 nM free Ca2+, pH 7.25, the mean membrane potential was -58 +/- 4.5 mV (n = 18). Cells were voltage clamped from -160 to +40 mV in 20-mV increments. The inward conductance was 767 +/- 221 pS/10 pF, and the outward conductance was 1,212 +/- 272 pS/10 pF (n = 12). Superfusion with 2 microM amiloride significantly hyperpolarized the cells by 7.4 +/- 2.2 mV (P = 0.004), significantly reduced the inward conductance to 221 +/- 65 pS/10 pF (P = 0.028), but had no effect on the outward conductance (1,294 +/- 236 pS/10 pF, P = 0.820, after amiloride). Next, the pipette and bath solutions were changed to (in mM) 150 sodium gluconate, 0.5 EGTA, and 20 HEPES, as well as < 10 nM free Ca2+, pH 7.25. Under these conditions amiloride significantly reduced (50%, P < 0.05; n = 7) the whole cell currents. When potassium gluconate was substituted for sodium gluconate, amiloride had no effect. Thus amiloride inhibited a Na(+)-specific conductance expressed by B lymphoid cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMed2000

Extracellular Cl(-) modulates shrinkage-induced activation of Na(+)/H(+) exchanger in rat mesangial cells.

To examine the effect of hyperosmolality on Na(+)/H(+) exchanger (NHE) activity in mesangial cells (MCs), we used a pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM, to measure intracellular pH (pH(i)) in a single MC from rat glomeruli. All the experiments were performed in CO(2)/HCO(-)(3)-free HEPES solutions. Exposure of MCs to hyperosmotic HEPES solutions (500 mosmol/kgH(2)O) treated with mannitol caused cell alkalinization. The hyperosmolality-induced cell alkalinization was inhibited by 100 microM ethylisopropylamiloride, a specific NHE inhibitor, and was dependent on extracellular Na(+). The hyperosmolality shifted the Na(+)-dependent acid extrusion rate vs. pH(i) by 0.15-0.3 pH units in the alkaline direction. Removal of extracellular Cl(-) by replacement with gluconate completely abolished the rate of cell alkalinization induced by hyperosmolality and inhibited the Na(+)-dependent acid extrusion rate, whereas, under isosmotic conditions, it caused no effect on Na(+)-dependent pH(i) recovery rate or Na(+)-dependent acid extrusion rate. The Cl(-)-dependent cell alkalinization rate under hyperosmotic conditions was partially inhibited by pretreatment with 5-nitro-2-(3-phenylpropylamino)benzoic acid, DIDS, and colchicine. We conclude: 1) in MCs, hyperosmolality activates NHE to cause cell alkalinization, 2) the acid extrusion rate via NHE is greater under hyperosmotic conditions than under isosmotic conditions at a wide range of pH(i), 3) the NHE activation under hyperosmotic conditions, but not under isosmotic conditions, requires extracellular Cl(-), and 4) the Cl(-)-dependent NHE activation under hyperosmotic conditions partly occurs via Cl(-) channel and microtubule-dependent processes.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed2006

A vH+-ATPase is present in cultured sheep ruminal epithelial cells.

In this study, the existence and functional activity of a vacuolar-type H(+)-ATPase (vH(+)-ATPase) was explored in primary cultures of sheep ruminal epithelial cells (REC). The mRNA transcripts of the E and B subunits of vH(+)-ATPase were detectable in RNA from REC samples by RT-PCR. Immunoblotting of REC protein extractions with antibodies directed against the B subunit of yeast vH(+)-ATPase revealed a protein band of the expected size (60 kDa). Using the fluorescent indicator BCECF and selective inhibitors (foliomycin, HOE 694, S3226), the contribution of vH(+)-ATPase and Na(+)/H(+) exchanger (NHE) subtype 1 and 3 activity to the regulation of intracellular pH (pH(i)) was determined in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium containing 20 mM of the short-chain fatty acid butyrate as well as after reduction of the extracellular Cl(-) concentration ([Cl(-)](e)) from 136 to 36 mM. The initial pH(i) of REC was 7.4 +/- 0.1 in nominally HCO(3)(-)-free, HEPES-buffered NaCl medium and 7.0 +/- 0.1 after acid loading with butyrate. Selective inhibition of the vH(+)-ATPase with foliomycin decreased pH(i) by 0.19 +/- 0.03 pH units. On the basis of the observed decreases in pH(i) resulting from inhibition of vH(+)-ATPase as well as of subtypes 1 and 3 of NHE, vH(+)-ATPase activity appears to account for approximately 30% of H(+) extrusion, whereas the activities of NHE subtypes 3 and 1 account for 20 and 50% of H(+) extrusion, respectively. Lowering of [Cl(-)](e) induced a pH(i) decrease (-0.51 +/- 0.03 pH units) and impaired pH(i) recovery from butyrate-induced acid load. Moreover, reduction of [Cl(-)](e) abolished the inhibitory effect of foliomycin and markedly reduced the HOE 694- and S3226-sensitive components of pH(i), indicating a role of Cl(-) in the function of these H(+) extrusion mechanisms. We conclude that a vH(+)-ATPase is expressed in ovine REC and plays a considerable role in the pH(i) regulation of these cells.

Amino Acid Sequence↗
PubMed2003

Anion-dependent Mg2+ influx and a role for a vacuolar H+-ATPase in sheep ruminal epithelial cells.

The K+-insensitive component of Mg2+ influx in primary culture of ruminal epithelial cells (REC) was examined by means of fluorescence techniques. The effects of extracellular anions, ruminal fermentation products, and transport inhibitors on the intracellular free Mg2+ concentration ([Mg2+]i), Mg2+ uptake, and intracellular pH were determined. Under control conditions (HEPES-buffered high-NaCl medium), the [Mg2+]i of REC increased from 0.56 +/- 0.14 to 0.76 +/- 0.06 mM, corresponding to a Mg2+ uptake rate of 15 microM/min. Exposure to butyrate did not affect Mg2+ uptake, but it was stimulated (by 84 +/- 19%) in the presence of CO2/HCO(-)3. In contrast, Mg2+ uptake was strongly diminished if REC were suspended in HCO(-)3-buffered high-KCl medium (22.3 +/- 4 microM/min) rather than in HEPES-buffered KCl medium (37.5 +/- 6 microM/min). After switching from high- to low-Cl- solution, [Mg2+]i was reduced from 0.64 +/- 0.09 to 0.32 +/- 0.16 mM and the CO2/HCO(-)3-stimulated Mg2+ uptake was completely inhibited. Bumetanide and furosemide blocked the rate of Mg2+ uptake by 64 and 40%, respectively. Specific blockers of vacuolar H+-ATPase reduced the [Mg2+]i (36%) and Mg2+ influx (38%) into REC. We interpret this data to mean that the K+-insensitive Mg2+ influx into REC is mediated by a cotransport of Mg2+ and Cl- and is energized by an H+-ATPase. The stimulation of Mg2+ transport by ruminal fermentation products may result from a modulation of the H+-ATPase activity.

Animals↗
PubMed1985

Sodium and chloride transport across rabbit ileal brush border. II. Evidence for Cl-HCO3 exchange and mechanism of coupling.

An inside-alkaline pH gradient (pH 7.7 inside, 5.5 outside) stimulated Cl uptake in brush-border vesicles from rabbit ileum. The addition of HCO3 without changing the pH gradient further stimulated Cl uptake to a level fourfold greater than equilibrated Cl uptake. Although a K diffusion potential stimulated Cl uptake, this was insensitive to inhibition by 4,4-diisothiocyanostilbene-2,2'-disulfonate (DIDS), whereas pH and HCO3 gradient-stimulated Cl uptake was inhibited by DIDS. pH and HCO3 gradient-stimulated Cl uptake was found to be a saturable function of the Cl concentration with a Km of 3.5 mM and a Vmax of 49 nmol X mg prot-1 X min-1. To distinguish between coupling of Na and Cl transport by cotransport or dual exchange (Na-H and Cl-HCO3 exchange), we determined uptake with high (134 mM Tris-HEPES-MES) internal buffer and low (1.34 mM Tris-HEPES-MES) internal buffer concentrations. Inwardly directed gradients of 50 mM Na, 50 mM K, or 50 mM Na and K did not stimulate Cl uptake, and 50 mM Cl, 50 mM K, or 50 mM KCl did not stimulate Na uptake, with high internal buffer, excluding cotransport. In contrast, 50 mM Na stimulated Cl uptake (inhibited by 1 mM DIDS) and 50 mM Cl stimulated Na uptake (inhibited by 1 mM amiloride) in low buffer media. To determine a role for carbonic anhydrase, Na-stimulated Cl uptake was determined in low buffer media, equilibrated with either 100% N2 or 95% N2-5% CO2. Na stimulated Cl uptake 80% (compared with trimethylammonium control) with CO2 but only 30% with N2 (P less than 0.05). Acetazolamide partially inhibited (P less than 0.025) the stimulation of Cl uptake with CO2 but not with N2. Carbonic anhydrase activity was measured in homogenate and brush-border membrane and was enriched 7.9 +/- 0.4-fold, whereas sucrase was enriched 14.0 +/- 1.1-fold. We conclude that coupled Na and Cl transport occurs by dual exchange (Na-H and Cl-HCO3) and carbonic anhydrase, apparently located on the brush-border membrane, facilitates dual exchange by providing HCO3.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗